The mechanism of cytochrome P-450-dependent oxidation of ethanol in reconstituted membrane vesicles.
The mechanism of cytochrome P-450-dependent oxidation of ethanol has been investigated using reconstituted phospholipid vesicles containing purified preparations of rabbit liver microsomal NADPH-cytochrome P-450 reductase and cytochrome P-450 LM2. Incorporation of cytochrome b5 into the vesicles resulted in a 5-fold enhancement of cytochrome P-450-catalyzed O-dealkylation of 7-ethoxycoumarin, whereas the cytochrome P-450-dependent ethanol oxidation was slightly inhibited. Superoxide dismutase, added in increasing amounts to the vesicles, inhibited the formation of superoxide anions and, in a concomitant manner, also the production of acetaldehyde from ethanol in the system. Also horseradish peroxidase inhibited ethanol oxidation catalyzed by the vesicles; acetaldehyde formation and H2O2 formation decreased in a concomitant manner as the amount of the peroxidase was increased. Externally added hydrogen peroxide markedly stimulated cytochrome P-450-dependent ethanol oxidation, but not until the concentration of H2O2 reached 0.3 mM, whereas the hydroxyl radical scavenger mannitol completely inhibited the cytochrome P-450-dependent acetaldehyde production. Oxidation of ethanol was also accomplished using vesicles containing cytochrome b5 instead of cytochrome P-450 and in other systems regenerating superoxide anions, e.g. the xanthine-xanthine oxidase system and dihydroxyfumarate. The results are consistent with an iron-catalyzed Haber-Weiss mechanism for regeneration of hydroxyl radicals which subsequently react with ethanol, thereby giving the corresponding aldehyde.
D000818:Animals; D003374:Coumarins / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D003580:Cytochromes / Q000494:pharmacology; D015786:Cytochromes b5; D000431:Ethanol / Q000378:metabolism*; D006735:Horseradish Peroxidase / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008081:Liposomes / Q000378:metabolism; D008353:Mannitol / Q000494:pharmacology; D008862:Microsomes, Liver / Q000201:enzymology; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D010084:Oxidation-Reduction; D011817:Rabbits; D013482:Superoxide Dismutase / Q000494:pharmacology
D003374:Coumarins; D003580:Cytochromes; D008081:Liposomes; C017299:7-ethoxycoumarin; D000431:Ethanol; D008353:Mannitol; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; D013482:Superoxide Dismutase; D009251:NADPH-Ferrihemoprotein Reductase